The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sweeney, S. E.
Right arrow Articles by Kim, Y. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sweeney, S. E.
Right arrow Articles by Kim, Y. B.
The Journal of Immunology, 2004, 172: 1203-1212.
Copyright © 2004 by The American Association of Immunologists

Identification of a Novel Fc{gamma}RIIIa{alpha}-Associated Molecule That Contains Significant Homology to Porcine Cathelin1

Susan E. Sweeney and Yoon B. Kim2

Department of Microbiology and Immunology, Finch University of Health Sciences/Chicago Medical School, North Chicago, IL 60064


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The following studies are the first to demonstrate the association of porcine Fc{gamma}RIIIa{alpha} with a molecule that contains significant homology to the cathelin family of antimicrobial proteins. We performed immunoprecipitation of the porcine Fc{gamma}RIIIa{alpha} multisubunit complex from Brij 96 lysates of polymorphonuclear leukocytes using the G7 mAb, which binds to Fc{gamma}RIIIa{alpha} on the surface of porcine NK cells and phagocytes. Previous results indicate that the transmembrane {alpha} subunit of the Fc{gamma}RIIIa complex is associated with the {gamma} subunit on the surface of porcine polymorphonuclear leukocytes and with several other unique proteins that surface iodinate and migrate at ~15, 20, and 25 kDa when analyzed by reducing SDS-PAGE. Through characterization of the porcine Fc{gamma}RIIIa complex, we identified the 15-kDa molecule as a unique Fc{gamma}R-associated protein that has not been described in other systems. We now report an association between Fc{gamma}RIIIa{alpha} and a 15-kDa molecule that shares homology to cathelin, a protein of undetermined function initially identified in porcine leukocytes. A domain with a high degree of homology to cathelin is found in the proregions of a family of antibiotic proteins referred to as cathelicidins. The results of our studies indicate the presence of a novel Fc{gamma}RIIIa complex in the porcine system, and may provide new insights into the function of this antimicrobial protein homologue in relation to the variety of responses mediated through Fc{gamma}Rs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Since the initial discovery of the cell surface receptors for the Fc region of Ig over 35 years ago, much new insight has been gained revealing the complexity and diversity of this group of molecules. FcRs, once regarded as distant relatives of the classical Ag receptors, are a critical coordinating element of the immune system. Receptors that bind to the Fc portion of IgG (Fc{gamma}R) link humoral and cellular immune responses and transform the interaction of Ag and Ab into a physiologically important event (1). FcRs are present on almost all cells of hemopoietic origin including NK cells, polymorphonuclear leukocytes (PMN),3 monocytes, macrophages, T cells, B cells, platelets, and nonhemopoietic cells such as endothelial cells (2, 3, 4). Receptor cross-linking results in the induction of a wide variety of responses that include phagocytosis, Ab-dependent cell-mediated cytotoxicity, release of reactive oxygen intermediates and lysosomal hydrolases, and synthesis and secretion of prostaglandins, leukotrienes, and other mediators of inflammation (2, 5, 6, 7, 8). The variety of responses evoked by the interaction of Ag-Ab complexes with Fc{gamma}Rs is a result of the extensive diversity present in this family of regulatory molecules. Fc{gamma}Rs have structurally similar ligand binding domains joined to distinct transmembrane and cytoplasmic domains. In addition, the formation of multisubunit receptors adds to the complexity of the Fc{gamma}R family and expands the variety of possible cellular responses that occur with the binding of immune complexes. Two classes of IgG FcRs are now recognized as activation or inhibitory receptors usually coexpressed on the cell surface. The balanced function of these receptors has been implicated in the maintenance of tolerance as well as the outcome of engagement of this receptor by the binding of IgG. These receptors mediate responses in a variety of systemic processes that include cellular differentiation, cytotoxicity, inflammation, autoimmunity, and allergy (1).

Three distinct but related classes of Fc{gamma}Rs (Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII) have been identified in both the human and murine systems. Classification is based mainly on biochemical properties of the extracellular Ig binding domain of these receptors (2). Human Fc{gamma}RIII (CD16), a low affinity Fc{gamma}R, is encoded by two homologous genes that generate a transmembrane molecule (Fc{gamma}RIIIa{alpha}) or a glycosyl phosphatidylinositol-linked molecule (Fc{gamma}RIIIb{alpha}) (9, 10). The human Fc{gamma}RIIIB gene is expressed as the glycosyl phosphatidylinositol-linked form of the receptor on PMN (Fc{gamma}RIIIb{alpha}) (10, 11, 12, 13, 14). The Fc{gamma}RIIIA gene encodes the transmembrane molecule (Fc{gamma}RIIIa{alpha}) found on human NK cells, macrophages, and subpopulations of T cells and cultured monocytes (2, 9, 15, 16, 17). Fc{gamma}RIIIa{alpha} forms a multimolecular complex consisting of the ligand binding {alpha} subunit in association with other subunit molecules such as Fc{epsilon}RI{gamma} (18, 19, 20) and TCR{zeta} (21, 22, 23). These disulfide-linked subunits associate as {gamma}-{gamma} or {zeta}-{zeta} homodimers, or as {gamma}-{zeta} heterodimers depending upon the cell type (19). Associated subunit molecules are important in both proper cell surface expression of the complex and signal transduction after ligation of the {alpha}-chain (18, 24). The Fc{gamma}RIIIa{alpha} molecule also associates with the {beta}-chain and a {gamma} homodimer on mast cells (25). Initially, many of these small associated molecules were not identified because they did not label efficiently with surface iodination.

The purpose of the following studies was to determine the composition of the porcine Fc{gamma}RIIIa multisubunit complex. We used the mild, nonionic detergent, Brij 96, in conjunction with the G7 mAb, which recognizes the ligand binding {alpha} subunit of the porcine Fc{gamma}RIIIa complex, to immunoprecipitate and characterize this cell surface complex (26). The G7 mAb binds to porcine Fc{gamma}RIIIa{alpha} and enhances NK-mediated and induces phagocyte-mediated cytotoxicity of FcR-positive tumor cells (27, 28, 29, 30). Porcine Fc{gamma}RIIIa{alpha} has been fully characterized and identified as an ~45-kDa glycoprotein (31). We have previously demonstrated that the transmembrane {alpha} subunit of the Fc{gamma}RIIIa complex is associated with the {gamma} subunit on the surface of porcine PMN, along with several unique proteins that migrate at ~15, 20, and 25 kDa by reducing SDS-PAGE (32). These unidentified proteins do not represent modified forms of the {gamma} or {zeta} subunits but each is labeled with cell surface iodination. We have now identified one of these unique Fc{gamma}R-associated proteins and have also demonstrated an association between Fc{gamma}RIIIa{alpha} and the 15-kDa protein that contains homology to cathelin, a protein of undefined function identified initially in porcine leukocytes (33). A cathelin domain is contained within the family of antimicrobial proteins known as cathelicidins. In addition to antimicrobial activity through permeabilization of cell membranes, many members of this family modulate inflammation and the Ag-driven immune response.

The results of our studies demonstrate the presence of a novel Fc{gamma}RIIIa complex in the porcine system, and may provide insight into the function of the Fc{gamma}RIIIa{alpha}-associated cathelin homologue in relation to the various responses mediated through Fc{gamma}Rs. Our model of the novel Fc{gamma}RIIIa complex on porcine PMN represents the first biological system characterized with the transmembrane {alpha} subunit of Fc{gamma}RIIIa present in association with a {gamma}-{gamma} homodimer on PMN. Because the unidentified associated molecules seem to be distinct from Fc{gamma}R-associated subunits described to date, these studies not only confirm the presence of a novel Fc{gamma}RIIIa molecular complex, but have also resulted in the identification of a unique Fc{gamma}R-associated molecule in the porcine system. Complete characterization of this novel Fc{gamma}RIIIa molecular complex identified in the porcine system (32) will undoubtedly yield valuable insights related to the structure and function of these immunologically significant receptors involved in cell-mediated cytotoxicity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and leukocyte preparation

Porcine PBMC were obtained from adult male and female specific pathogen-free (SPF) Minnesota miniature swine of various ages maintained at Finch University of Health Sciences/Chicago Medical School (North Chicago, IL) in barrier-sustained SPF facilities. Peripheral blood was aseptically collected using a 13–15 gauge needle and a 60-cc syringe by cardiac puncture of animals anesthetized with ketamine HCl (Fort Dodge Laboratories, Fort Dodge, IA) and CO2. The blood was transferred to a sterile bottle that contained 10 U/ml sodium heparin. Porcine PBMC were isolated by Ficoll-Hypaque density centrifugation as previously described (34). PBMC were collected from the interface of the plasma and Ficoll-Hypaque solution and were washed three times with PBS. PBL were isolated by depletion of adherent monocytes as described previously (31).

PMN were prepared by dextran sedimentation following Ficoll-Hypaque separation of whole blood as described previously (29). A modified large scale method of isolation of porcine PMN was used for large volumes of blood, which essentially involves performing the dextran sedimentation before the Ficoll-Hypaque gradient was performed as described previously (26). Porcine pulmonary alveolar macrophages (PAM) were isolated from adult Minnesota miniature swine by lavage using ice-cold PBS as described previously (35).

Antibodies

The G7 mAb was produced as previously described (30). MOPC-31c (Sigma-Aldrich, St. Louis, MO) was used as an IgG1 isotype control. Rabbit anti-pig proprotegrin (anti-PPG) serum was generously supplied by Drs. T. Ganz and J. Shi (University of California Los Angeles Medical Center, Los Angeles, CA). Guinea pig anti-rabbit p15 serum was a generous gift from Dr. P. Elsbach (New York University School of Medicine, New York, NY). Rabbit anti-human {gamma} serum was kindly provided by Dr. J. V. Ravetch (Rockefeller University, New York, NY).

Large scale immunoprecipitation of the G7 Brij 96 complex

The large scale immunoprecipitation was performed as described previously with the following modifications (26). Ten milligrams of mouse IgG (Sigma-Aldrich) or 10 ml of G7 ascites were conjugated to 2 and 6 ml, respectively, of protein G-agarose beads overnight at 4°C. The Ab-bead conjugates were washed eight times with PBS and two times with 10 volumes of sodium borate buffer, pH 9.0. The Ab was then covalently linked to the protein G beads using dimethylpimeliminidate (DMP). Approximately 1011 PMN were isolated from porcine blood using the large scale procedure as described in the leukocyte preparation section of Materials and Methods. An aliquot of ~108 cells was removed for surface iodination followed by immunoprecipitation. The remaining unlabeled cells were lysed immediately in ice-cold Brij 96 lysis buffer, containing protease inhibitors, at 108 cells/ml with mixing for 45 min on ice. The lysate was then centrifuged at 12,000 x g for 30 min at 4°C. The supernatant was collected and precleared overnight at 4°C using the mouse IgG cross-linked protein G beads. A second preclear step was performed using protein G-agarose beads alone for 4 h at 4°C. The precleared lysate was then incubated with the G7 mAb cross-linked beads overnight at 4°C to immunoprecipitate the Brij 96 complex. The following day, the beads were washed with Brij 96 lysis buffer containing protease inhibitors. The Brij 96 G7-associated proteins were isolated by addition of 200 µl of PBS, containing 0.1% SDS, followed by heating at 100°C for 5 min to batch elute the complex. The eluted protein was collected and this procedure was repeated twice. The eluates were combined and concentrated in a Centricon 3 concentrator (Amicon, Beverly, MA) to a minimum volume of ~25 µl. The retentate was then dried in a vacuum centrifuge until completely dry. The pellet was washed twice with 1 ml of MeOH, centrifuged at 12,000 x g for 20 min at 4°C, and allowed to air dry. This procedure was necessary to remove any remaining Brij 96 detergent that forms large aggregates on heating at 100°C and becomes concentrated with the protein sample of interest. This concentrated detergent aggregate will then interfere with analysis by SDS-PAGE if not removed with a MeOH wash of the dried sample. The pellet was resuspended in reducing sample buffer and solubilized with brief heating. The immunoprecipitated G7 complex was then analyzed by reducing SDS-PAGE on a 15% gel. Samples of a precleared as well as a direct G7 immunoprecipitation of the iodinated complex were included on the same gel with the unlabeled large scale G7 mAb immunoprecipitate to align with the proteins of interest. The separated proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane that was sealed wet in plastic and exposed to film overnight at -70°C. The membrane was stained with Ponceau S stain (0.2% Ponceau S in 1% acetic acid) followed directly by amido black stain (0.1% amido black in 1% acetic acid and 40% MeOH) for an increased level of sensitivity of protein detection. The membrane was rinsed in reagent grade double-distilled H2O followed by excision of the bands of interest. Each band was then washed five times with reagent grade double-distilled H2O with 5% MeOH in a microfuge tube.

Microsequence analysis of the 15-kDa Fc{gamma}RIIIa{alpha} associated molecule

A portion of the digestion, separation, and amino acid analysis was performed by S. Latshaw in the Protein Sequencing Laboratory (Department of Biological Chemistry, Finch University of Health Sciences/Chicago Medical School). For samples analyzed at Finch University of Health Sciences/Chicago Medical School, a PVDF tryptic digest with reduced Triton X-100 (RTX-100) was performed and the resulting peptides were separated using a Vydac-201TP5414 (4.6 mm x 150 mm) reverse phase column on a Maxima 820 HPLC (Hesperia, CA). The remaining digestion, separation, and sequence analyses, as well as all of the mass spectrometry were performed by W. S. Lane, R. Robinson, J. Neveu, and E. Spooner of the Harvard Microchemistry Facility (Harvard University, Cambridge, MA). For samples sent to Harvard Microchemistry, in situ digestion was performed with endoprotease Lys-C, a variation of the classic trypsin method (36). A small portion of the sample was used for amino acid analysis and the rest of the peptide mixture was separated by narrow-bore high performance liquid chromatography using a ZORBAX C18 (1 mm x 150 mm) reverse-phase column on a Hewlett-Packard Protein Sequencer with Online 1090 High Performance Liquid Chromatograph (Palo Alto, CA). Optimum fractions from the chromatogram were chosen with regard to peak symmetry and peak width (resolution) as well as UV absorption at 205, 277, and 292 nm. In addition, the molecular mass and peptide purity within selected fractions were assessed at Harvard Microchemistry using matrix-assisted laser desorption time-of-flight mass spectrometry performed on a Finnegan Lasermat 2000 (Hemel, U.K.). Selected fractions were subjected to internal amino acid sequencing using automated Edman degradation on an Applied Biosystems 477A protein sequencer (Foster City, CA) with a 120A PTH-AA analyzer. The sequences obtained were compared with known sequences using the PC/GENE (Intelligenetics, Mountain View, CA) program and the SWISS/PROT database.

Radioiodination, immunoprecipitation, and analysis by SDS-PAGE

Cells were surface labeled with 125I using a modified lactoperoxidase method (37) which has been described previously (27). Cells were washed and then lysed in Brij 96 (Sigma-Aldrich) lysis buffer (1% Brij 96, 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin) on ice for 45 min. Nuclei were removed by centrifugation at 12,000 x g for 15 min at 4°C. Cell lysates were precleared using 100 µl of protein G-agarose beads (Boehringer Mannheim, Indianapolis, IN) and 50 µg of mouse IgG (Sigma-Aldrich) or normal rabbit serum and overnight rotation at 4°C. Abs to be used for immunoprecipitation were preconjugated to protein G-agarose beads overnight at 4°C followed by covalent cross-linking of the Fc portion of the Ab to the beads using DMP (Sigma-Aldrich) (38). The Ab-conjugated beads were washed twice with 10 volumes of 0.15 M sodium borate, pH 9.0. The beads were then resuspended to 10 volumes and solid DMP was added to 20 mM. The mixture was rotated at room temperature for 2 h followed by quenching with 0.2 M ethanolamine, pH 8.0, for 2 h at room temperature. cross-linked Ab-bead conjugates were washed twice with PBS. Precleared supernatants were then rotated for 2 h at 4°C with 80 µl of protein G-agarose cross-linked to 10 µg of purified mAb (MOPC-31c or G7 mAb) or 5 µl of polyclonal antiserum (rabbit anti-PPG, rabbit anti-{gamma}, or guinea pig anti-rabbit p15). Ab-bead conjugates were washed five times in ice-cold lysis buffer. Immunoprecipitated proteins were eluted by heating at 100°C in sample buffer followed by SDS-PAGE on a 15% gel under reducing conditions (39). After electrophoresis, the gel was dried and exposed to film with an intensifying screen at -70°C for up to 1 wk.

Western blot analysis

MOPC-31c mAb, G7 mAb, normal rabbit serum, rabbit anti-PPG serum, and rabbit anti-{gamma} serum were used for immunoprecipitation from precleared PMN lysates prepared in 1% Brij 96 lysis buffer. Immunoprecipitated proteins were then separated by SDS-PAGE on a 15% reducing gel, transferred to a PVDF membrane (Bio-Rad, Hercules, CA), and rinsed in PBS. Nonspecific protein binding sites were blocked by incubation with 5% Carnation nonfat dry milk and 0.1% azide in PBS for 2 h at room temperature with rocking. The blot was then sealed in a MicroSeal bag (Dazey, Industrial Airport, KS) containing a 1/50 dilution of anti-PPG serum in blocking buffer overnight at 4°C with rocking. The blots were removed and washed with 0.1% Tween 20 in PBS. A 1/500 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich) was then prepared in blocking buffer. The blots were incubated at room temperature for 1 h with rocking and washed extensively in 0.1% Tween 20 in PBS at room temperature with rocking. The blots were developed using the Western-Light Plus Chemiluminescent Detection System (Tropix, Bedford, MA) as indicated by the manufacturer.

Flow cytometric analysis

Porcine PMN were washed with PBS, counted, and adjusted to a concentration of 1 x 107/ml. One hundred microliters of cells were aliquoted to tubes and a saturating amount of MOPC-31c ascites (IgG1 isotype control), G7 ascites, normal rabbit serum (NRS), or anti-PPG serum, ~4 µl, was added to each tube. After mixing, the tubes were incubated on ice for 45 min. The cells were then washed twice with 3 ml of cold PBS. Approximately 5 µg of secondary reagent, either FITC-conjugated affinity purified F(ab')2 goat anti-mouse IgG or FITC-conjugated affinity purified F(ab')2 goat anti-rabbit IgG (Organon Teknika, West Chester, PA), were then added to each tube, which were mixed by vortexing, and then incubated in the dark on ice for 30 min. Samples were then washed twice with ice-cold PBS, and once with 3% formaldehyde in PBS to fix the cells. Samples were stored in the dark at 4°C until analysis. Negative controls included cells stained with the IgG1 isotype control, MOPC-31c ascites, or with NRS followed by the appropriate secondary reagent.

All samples were analyzed on a Coulter ELITE ESP flow cytometer (Hialeah, FL) using an argon laser at 488 nm. The cell population of interest was mapped and gated based on size (forward light scattering) and granularity (90° light scattering). The gated population was then analyzed for fluorescent intensity and percent positive events. Specific positive staining with the G7 mAb or anti-PPG serum is insured by gating to subtract nonspecific staining with MOPC or NRS negative control, respectively, from the test samples. For all samples, events were recorded on a log fluorescence scale and fluorescence intensity data was generated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Large scale immunoprecipitation of the Fc{gamma}RIIIa{alpha} Brij 96 complex for microsequence analysis of associated molecules

As outlined in Materials and Methods, the porcine Fc{gamma}RIIIa complex was immunoprecipitated from Brij 96 lysate of ~1011 unlabelled porcine PMN using the G7 mAb (Fig. 1, left lane). In addition, a small scale immunoprecipitation from 108 surface iodinated PMN was performed from the same batch of cells also using the G7 mAb (Fig. 1: right lanes, lane 1 = standard preclear, lane 2 = no preclear). The membrane was exposed to x-ray film to allow alignment of the 125I-labeled proteins with the proteins in the unlabeled large scale immunoprecipitation lane on the left. The large scale G7 immunoprecipitation lane (G7) shows the positions of the excised bands and their relative alignment with the 125I-labeled G7-associated proteins (Fig. 1). Excised bands were stored at -20°C before sending for sequence analysis. We began characterization of the G7-associated proteins with the 15-kDa molecule.



View larger version (94K):
[in this window]
[in a new window]
  		FIGURE 1. Large scale immunoprecipitation of the G7 Brij 96 complex for amino acid sequencing of G7-associated molecules. The left lane contains proteins from the large scale immunoprecipitation from Brij 96 lysate of 1011 porcine PMN using the G7 mAb. This lane (large scale-G7) represents the amido black stained PVDF membrane after excision of the various bands of interest. The lanes on the right are autoradiograms from the same membrane of the G7 mAb immunoprecipitated Brij 96 complex from a portion (108) of the total PMN (1011) which were surface iodinated to serve as an alignment marker for G7-associated molecules (125I ippt-G7). Lane 1, An immunoprecipitation using the G7 mAb following a preclear step; lane 2, an immunoprecipitation using the G7 mAb without a preclear step. Samples were immunoprecipitated and analyzed as outlined in Materials and Methods.

 
Results of microsequence analysis of the G7-associated protein that aligns with the 15-kDa iodinated protein

Digestion, separation, and amino acid sequencing of the 15-kDa protein was performed on two independent large scale immunoprecipitations of the G7 Brij 96 complex. One complete digestion, separation, and sequencing procedure was performed at Finch University of Health Sciences/Chicago Medical School. Peak number 61 from the HPLC profile of the proteolytic digest with trypsin performed at Finch University of Health Sciences/Chicago Medical School was subjected to amino acid sequence analysis. In addition, the 15-kDa protein PVDF sample from an independent purification was subjected to digestion, separation, mass spectroscopic analysis, and amino acid sequence analysis at Harvard Microchemistry. The HPLC profile of this endoprotease Lys-C digest was performed at Harvard Microchemistry. Peaks 11, 69, and 84 were chosen for analysis on the basis of UV absorption, peak symmetry, and peak resolution. The mass spectroscopic analyses of these fractions was performed by Harvard Microchemistry to estimate the peptide purity and molecular mass of the selected HPLC peaks using matrix-assisted laser desorption time-of-flight mass spectrometry on a Lasermat as described in Materials and Methods (data not shown).

Peaks 11, 69, and 61 resulted in high confidence amino acid sequences. The amino acid sequence results of the three peaks from which the high confidence sequences were obtained (PK11, PK69, and PK61) are presented in Table I. These sequences were subjected to homology searches of the SWISS PROT database using the FASTA program of PC/GENE. The results of these homology searches are also shown in Table I. The first peptide sequence obtained from the 15-kDa protein resulted from sequence analysis of peak 11 (PK11) of the HPLC profile performed at Harvard Microchemistry. The high confidence sequence results consisted of the amino acids PVSFTVK. Using this information alone, the top three proteins, porcine cathelin, bovine indolicidin, and rabbit CAP18, were a 100% match to this seven amino acid stretch. To further confirm the identity of the 15-kDa G7-associated protein, additional sequence data from the 15-kDa protein was obtained from peak 69 (PK69) of the HPLC profile from Harvard Microchemistry. The sequence VLRAVDXL, with X representing any amino acid, was scanned against all the protein sequences in the protein database. The same cathelin/cathelicidin family of molecules including cathelin, bactenecin, indolicidin, and CAP18 were included in the top 25 proteins to match this partial sequence ranging from 75 to 88% match in the region of overlap (Table I). We then used the CLUSTAL program of PC/GENE to perform a multiple sequence alignment of the proteins cathelin, bactenecin, indolicidin, and CAP18. It was evident from this alignment that both of the sequences, PVSFTVK and VLRAVDXL, were highly conserved within this family of proteins. An additional piece of sequence data of the 15-kDa protein obtained from peak 61 (PK61) of the HPLC profile from the Finch University of Health Sciences/Chicago Medical School protein sequence laboratory consisted of the sequence LLELDQPPKADEDPGTPK. The results of the homology search indicated that these 18 amino acids were a 100% match for a region of the porcine cathelin sequence (Table I). In addition, this region of sequence showed less homology, 68–78%, to the other members of this family, including bactenecin and indolicidin, which are from different species. The rabbit CAP18 protein did not score in the top 25 proteins retrieved from the homology search. Therefore, this particular sequence allowed the identity of the 15-kDa protein to be narrowed more specifically to porcine cathelin or a porcine cathelicidin.


View this table:
[in this window]
[in a new window]
  		Table I. Amino acid sequence results of proteolytic digest peptides from the 15-kDa Fc{gamma}RIIIA-associated molecule

 
All three of the digest peptide sequences of the 15-kDa protein are present in the amino acid sequence of a molecule known as porcine cathelin. The published amino acid sequence of porcine cathelin is shown in Fig. 2, with tryptic digest peptides underlined. The three digest peptides demonstrate 100% homology to the cathelin protein. These sequences are also present in the N-terminal region of the proforms of members of the cathelicidin family of antibiotic proteins (40). Multiple protein sequence alignment of homologous members of the cathelicidin family of microbicidal proteins is shown in Fig. 3. As would be expected, the peptide sequences also have counterparts in the sequences of PR-39, PPGs, porcine myeloid antibacterial peptides (PMAPs), and prophenins, all of which contain a conserved cathelin domain. The function of the cathelin protein itself is unknown, but the proforms of members of the cathelicidin family are processed to release a C-terminal cationic antibacterial peptide from the cathelin-like proregion (41). From this data, we concluded that the 15-kDa Fc{gamma}RIIIa{alpha}-associated protein contains significant sequence homology to the porcine cathelin/cathelicidin family.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 2. Amino acid sequence analysis of the 15-kDa Fc{gamma}RIIIa{alpha}-associated subunit indicates significant sequence homology to porcine cathelin. The published sequence of the cathelin protein is used in this figure to demonstrate alignment of this protein with the proteolytic digest peptides of the 15-kDa subunit of the Fc{gamma}RIIIa{alpha} complex. Digest peptides are indicated by an arrow followed by the underlined peptide fragment sequence. These three peptides are 100% homologous to the porcine cathelin protein.

 


View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 3. Multiple protein sequence alignment of homologous members of the cathelicidin family of microbicidal proteins. Sequences homologous to proteolytic digest peptides are shaded; actual peptide sequence from the 15-kDa molecule is underlined. Arrows indicate digest sites. BCT, bactenecin; INDC, indolicidin; PG, protegrin; PRN, prophenin; PR, proline arginine-rich antimicrobial protein; FALL-39, LL-37.

 
Confirmation of the association of a 15-kDa cathelin homologue with porcine Fc{gamma}RIIIa{alpha}

To confirm association of Fc{gamma}RIIIa{alpha} with the 15-kDa cathelin-like protein found in our system, we needed an Ab to the porcine cathelin protein or the conserved cathelin domain in the precursor forms of any of the members of the cathelicidin family. We received two different Abs, namely rabbit polyclonal anti-pig PPG serum and guinea pig anti-rabbit p15 serum. The results of immunoprecipitation with anti-PPG are similar to that with the G7 mAb, with both Abs immunoprecipitating the Fc{gamma}RIIIa{alpha} protein at 40 kDa as well as the other Brij 96-associated proteins at 15 and 20 kDa from the surface-iodinated PMN (Fig. 4). The anti-p15 serum appears to weakly immunoprecipitate the G7 Brij 96 complex. This complex is not present in the negative control immunoprecipitation lanes, MOPC and NRS, confirming the specificity of the association between members of this complex. We used the anti-pig PPG serum to further investigate the association of porcine Fc{gamma}RIIIa{alpha} with a cathelin-like protein.



View larger version (135K):
[in this window]
[in a new window]
  		FIGURE 4. Immunoprecipitation of the G7 complex from Brij 96 cell lysates of surface iodinated PMN using mAbs or antisera directed against members of the cathelicidin family of molecules which may associate with the porcine Fc{gamma}RIIIa complex. MOPC-31c (mouse IgG1 isotype control), NRS, and NGPS (normal guinea pig serum) are included as negative controls. PPG, anti-PPG (recognizes a conserved cathelin domain in the precursor protein); anti-p15, guinea pig serum raised against the rabbit 15-kDa antibacterial protein. Samples were analyzed by reducing SDS-PAGE on a 15% gel. This figure is representative of two experiments.

 
To confirm an association between Fc{gamma}RIIIa{alpha} and a cathelin homologue, we performed Western blot analysis of the immunoprecipitated Brij 96 complex using anti-PPG as the primary Ab (Fig. 5). The anti-PPG serum reacts with a protein at ~12–15 kDa in both the G7 mAb and anti-PPG immunoprecipitation lanes. This 12–15 kDa band is not present in the MOPC or NRS negative control immunoprecipitation lanes indicating that the PPG protein specifically associates with porcine Fc{gamma}RIIIa{alpha}. The anti-{gamma} immunoprecipitation lane in Fig. 5 may contain a very faint band present at the same Mr. The anti-PPG serum immunoprecipitates quantitatively more of the PPG protein as compared with the G7 mAb immunoprecipitation. This may indicate that only a portion of the PPG/cathelin homologues in the cell are associated with the G7 complex on the cell surface. Alternatively, this observation may be the result of a difference in affinity of each Ab for their respective target Ags. It is also possible that the efficiency of the G7 mAb to coprecipitate the cathelin homologue may differ from the direct ability of the anti-PPG to immunoprecipitate the cathelin-like protein. The ability of the Brij 96 detergent to maintain the noncovalent associations within the complex represents an additional explanation for this quantitative difference in immunoprecipitation of protein. In the lanes labeled PMN and PAM, the cell lysate supernatant was loaded directly onto the corresponding lanes of the same gel analyzed by reducing SDS-PAGE. The anti-PPG serum recognized three different proteins in cell lysate of porcine PMN, the smallest of which aligned with the single band in both G7 mAb and anti-PPG immunoprecipitations. Because the lysate lanes contain all the cathelicidins expressed in that particular cell type, several bands ranging from ~10 to 18 kDa would be expected to be present in the PMN lysate. These bands represent various members of the cathelicidin family expressed in that particular cell type that contain the cathelin domain recognized by the anti-PPG serum. The smallest members of the porcine cathelicidin family include cathelin and the PPGs. With respect to size, the results shown in Fig. 5 indicate that the specific cathelin homologue immunoprecipitated with G7 mAb aligns with the smaller members of the cathelicidin family. The protein homologous to cathelin that associates with Fc{gamma}RIIIa{alpha} may represent one of the PPGs or the cathelin protein. The results of the lysate lanes in Fig. 5 also suggest that the levels of these cathelin family proteins may differ depending on the cell type studied. In addition, this study demonstrates that there are variable sizes of cathelin homologues in these cell types that do not associate with Fc{gamma}RIIIa{alpha} and are not coprecipitated using anti-Fc{gamma}RIIIa{alpha} mAb and the Brij 96 cell lysate. This is additional evidence to support the specificity of the association of the 15-kDa cathelin homologue with Fc{gamma}RIIIa{alpha}.



View larger version (97K):
[in this window]
[in a new window]
  		FIGURE 5. Western blot analysis of G7 mAb, anti-PPG, or anti-{gamma} immunoprecipitated complex from Brij 96 lysate of porcine PMN using polyclonal rabbit anti-PPG serum as the primary Ab. MOPC immunoprecipitates are included as a negative control for the G7 mAb. NRS immunoprecipitates are included as a negative control for the anti-PPG and anti-{gamma} immunoprecipitations, respectively. In the lanes labeled PMN and PAM, cell lysate was loaded directly in the corresponding lanes on the right. PMN, PMN lysate lane; PAM, PAM lysate lane. Samples were separated by reducing SDS-PAGE on a 15% gel. This figure is representative of three experiments.

 
Because porcine PMN lysate contains several cathelicidins and quantitatively much higher levels of these proteins as compared with PAM, it was possible that the cell type expression of these cathelin homologues may be similar to that of the G7 protein, with highest amounts on PMN, intermediate amounts on PAM, and lowest levels on PBL representing the percentage of these cells which are NK cells and are FcR positive. Data from Western blot experiments performed with equal amounts (106 per lane) of PMN, PAM, and PBL lysates suggested that the cathelin homologue was expressed at quantitatively higher levels in PMN, an intermediate level in PAM, and practically undetectable levels in PBL (data not shown). It was possible that this initial negative result was due to the fact that the T cells and B cells that make up 90% of porcine PBL would not be expected to contribute to the anti-PPG reactivity. If the positive population did indeed represent the 10% of PBL that are NK cells, we expected that increasing the amount of PBL lysate could result in detection of this protein. This experiment was then repeated using excess PBL and an increased number of PAM to achieve a detectable signal in all lanes (Fig. 6). The PBL lane, although overloaded with protein, contains a band at the approximate Mr of the cathelin homologue as well as other members of the cathelin family that would be expected to react with the anti-PPG serum. The larger members of the cathelicidins are more evident in the PAM and PMN lanes and migrate from 10 to 18 kDa confirming the results in Fig. 5. The results in Fig. 6 suggest that a cathelicidin-like molecule is expressed in all three of these porcine cell types that also express Fc{gamma}RIIIa{alpha}. The cell populations will be immunostained with anti-PPG to determine the percent positive as well as the purity of the isolated cell populations. The possibility of false-positive PPG staining due to contaminating cell populations, such as an average of 3% PMN obtained from BAL for PAM of SPF adult pigs, cannot be excluded at this point in time. Several high Mr proteins are present in these Western blot experiments probably due to the use of cell lysate in combination with the unpurified rabbit antiserum. The bands are not present in the experiments in which the G7 or PPG complex is specifically immunoprecipitated followed by Western blot analysis of the complex (Fig. 5) suggesting that the bands are the result of cross-reactivity of the antiserum with the numerous proteins present in cell lysate. Overall, this data suggests the presence of a cathelin homologue on these additional cell types, however, the composition of the complex on porcine PAM and PBL (NK cells) has not yet been studied. Therefore, the presence of a specific association between a cathelin homologue and the Fc{gamma}Rs on these cell types remains undetermined at the present time. However, the cell-specific level of expression of the cathelin homologue may indeed be similar to that of Fc{gamma}RIIIa{alpha}.



View larger version (103K):
[in this window]
[in a new window]
  		FIGURE 6. Western blot analysis of cell lysate of porcine PBL, PAM, and PMN using polyclonal rabbit anti-PPG serum as the primary Ab. Samples were loaded unequally to achieve relatively similar reactivity in these three cell types. Approximately 4 x 107 PBL, 107 PAM, and 5 x 105 PMN were lysed directly in reducing sample buffer. Samples were separated by SDS-PAGE on a 15% reducing gel, transferred to a PVDF membrane, and blotted using the anti-PPG serum as the primary Ab. This figure is representative of four experiments.

 
Because the literature indicated that cathelicidins were mainly localized to intracellular granules, we performed cell surface staining using the anti-PPG serum as well as the G7 mAb as a positive control. The results of surface staining of porcine PMN using the G7 mAb or anti-PPG are presented in Fig. 7. As expected, PMN are ~99% positive when stained with the G7 mAb. Surprisingly, ~93% of the PMN are also surface positive with the anti-PPG staining. To eliminate the possibility that the PMN were degranulating during the isolation procedure and releasing a cathelin-like molecule that subsequently bound nonspecifically to the cell surface, a staining of fresh whole blood was also performed and resulted in positive surface staining results. Gating cell populations after RBC lysis revealed similar percent positive PPG as expected for Fc{gamma}RIIIa{alpha} on PBL and PMN (data not shown). Therefore, we conclude that on porcine PMN a protein with significant sequence homology to cathelin and the cathelicidin family of proteins is expressed on the cell surface in association with Fc{gamma}RIIIa{alpha}.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 7. Flow cytometric analysis of surface expression on porcine PMN surface stained with the G7 mAb or polyclonal anti-PPG serum. MOPC and NRS are included as negative controls. Specific positive staining with the G7 mAb or anti-PPG serum is obtained by gating only on the positive population as compared with the nonspecific staining with MOPC and NRS controls, respectively. This figure is representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To further characterize the porcine Fc{gamma}RIIIa complex, we performed large scale immunoprecipitation of the complex, and sequence results from the 15-kDa protein provided the first important clue as to the nature of this Fc{gamma}RIIIa{alpha}-associated protein. Table I summarizes the results from peptide sequencing of the 15-kDa protein and homology searches of a protein database. It is evident that this molecule shares significant sequence identity to the family of molecules referred to as cathelicidins. Due to the high degree of homology of the proregion of this family of molecules to cathelin, a 12-kDa protein isolated from porcine PMN, this antimicrobial family of proteins was named cathelicidins (40). The general cathelicidin structure includes an N-terminal signal sequence preceded by a negatively charged cathelin homologous proregion that is followed by a positively charged C-terminal antibacterial peptide. The C-terminal peptide is released in mature form after proteolytic processing and is highly variable between members of this same family (41). Initially, cathelin was identified as a cysteine protease inhibitor but this activity was later attributed to a protein which was inadvertently copurified with cathelin (33). The function of the proregion within the cathelicidins has been theorized to neutralize the charged C-terminal portion to prevent antibiotic effects of this region before processing, target the protein to the appropriate organelle, assist in folding of the mature protein, or serve a novel function yet to be identified (42). Recently, overexpression and crystal structure study of the cathelicidin motif of the protegrin-3 precursor revealed a monomeric and domain-swapped dimeric structure. This structure has been proposed to allow modulation of protein activity and alter the secretion, trafficking, and storage of these bactericidal, membrane-disruptive peptides (57, 58). A multiple sequence alignment of some of the representative members of this protein family is shown in Fig. 3. The actual sequences of peptides analyzed from the proteolytic digest of the 15-kDa Fc{gamma}RIIIa{alpha}-associated protein are underlined. Sequences that match portions of the peptide are shown in shaded areas. It is clear that the 15-kDa G7-associated molecule could be any one of the many porcine antibacterial proteins. Porcine cathelin homologues include the proforms of several antibiotic peptides produced by porcine leukocytes including the protegrins, which share some homology to the defensin family of antimicrobial proteins (42, 43) and exhibit broad antimicrobial activity (44, 45). PR-39 is a proline- and arginine-rich porcine antimicrobial peptide which has recently been proposed to serve an additional role in the healing process of wounds (46). The PMAP (47) and the phenylalanine-rich prophenins (48) were identified as peptides or by using PCR and primers based on the conserved cathelin sequence of the proregions.

Rabbit serum specific for the propiece of PPG, which is also known as the cathelin domain, was used to confirm the molecular association. The anti-PPG serum coprecipitates the 40-kDa Fc{gamma}RIIIa{alpha} molecule from surface-iodinated PMN. In an effort to further demonstrate the specificity of the very unique association between the ligand binding {alpha} subunit of the porcine Fc{gamma}RIIIa complex and a porcine cathelicidin, we performed immunoprecipitation followed by Western blot analysis. This data demonstrates a unique association between the Fc{gamma}RIIIa{alpha} protein and a cathelin-like molecule on porcine PMN. It is not possible to draw conclusions regarding a specific association between the {gamma}-chain and the cathelin-like molecule because this has not been directly demonstrated experimentally. Future studies will investigate the existence of this association because we have previously demonstrated that the Fc{gamma}RIIIa{alpha} and a {gamma}-{gamma} homodimer are members of the Brij 96 complex on porcine PMN and have published these results (32). The stoichiometry between Fc{gamma}RIIIa{alpha} and the cathelin homologue appears to be 1:1 on the cell surface from SDS-PAGE analysis of PMN. Western blot experiments also indicate that the anti-PPG serum may react with proteins from lysates of both porcine PMN and PAM. The smallest of the three proteins aligns with the band reactive with anti-PPG in the G7 mAb and anti-PPG immunoprecipitates. Based on predicted size, the protein probably represents cathelin or one of the protegrins. We then performed Western blot analysis on lysates of porcine PMN, PAM, or PBL to obtain a complete picture of cell type expression of this cathelin-like molecule. Initial experiments in which equal amounts of cell lysate were loaded into each lane resulted in negligible reactivity of the anti-PPG serum with the PBL lysate. Because T cells and B cells represent ~90% of porcine PBL, this result suggested that these cells probably do not express the cathelin family proteins and serve as cathelin-negative cell populations. By greatly increasing the amount of PBL cell lysate used, cathelin reactivity was evident in this cell lysate, possibly representing the 10% of the population that consists of NK cells. The results of these experiments suggested that the cathelicidin-like molecule is likely expressed in all three of these porcine cell types. Previous reports of G7 expression in the porcine system indicated that this protein is expressed on the surface of the same cell types in similar relative amounts. Cell-specific double staining experiments will be performed to determine whether the cathelicidin surface expression truly parallels that of Fc{gamma}RIIIa{alpha}.

Next we performed experiments using flow cytometry to investigate surface expression of a cathelin-like molecule on porcine leukocytes. Because the literature indicated that the proforms of most of the cathelicidins were stored in intracellular granules of PMN (49, 50), we were initially skeptical not only of this unexpected association involving Fc{gamma}RIIIa{alpha}, but also of localization to the cell surface. At the present time, the localization as well as the function of the porcine cathelin protein is unknown. The localization of many of the cathelicidins seemed to be restricted mainly to intracellular granules of PMN, but some have been found in the extracellular environment. Bovine probactenecins have been shown to be released from in vitro-stimulated PMN (49). Additionally, porcine PR-39 and the rabbit p15s have also been shown to be released into the extracellular environment (46, 51). Localization of members of the cathelicidin family to inflammatory exudates suggests that these proteins may serve additional extracellular functions (41). Localization in other species to the secondary granules of PMN, which are more readily mobilized to release their contents extracellularly, has also given support to the theory that this family of proteins may serve an important extracellular function. The human antibacterial cathelicidin hCAP-18 has been demonstrated to be bound to lipoproteins in plasma which is mediated by the C-terminal antibacterial portion of the protein (52). However, none of these cathelin-like proteins has been demonstrated to exist on the cell surface. Flow cytometric analysis was performed to investigate whether a molecule with cathelin homology could be expressed on the surface of porcine leukocytes. We performed standard isolation of PMN followed by surface staining with rabbit anti-PPG serum. In our system, nonpermeabilized PMN stained positive with anti-PPG serum as compared with the NRS control. The staining pattern was similar to that of Fc{gamma}RIIIa{alpha} and confirmed the surface expression of a cathelin homologue on this cell type. In addition, surface membrane staining with the anti-PPG serum was performed on PMN, PAM, and PBL (data not shown). All three cell types showed positive surface staining with anti-PPG as detected by fluorescence microscopy.

These results, though encouraging, were subject to the criticism that inadvertent cellular activation during the isolation procedure caused the release of the cathelin-like molecule and subsequent nonspecific binding to the cell surface, which could then be detected by surface staining and flow cytometric analysis. To address this issue, we performed surface staining directly on fresh whole blood using the anti-PPG serum. By gating specific cell populations, we found that PMN did stain >90% surface positive and PBL ~10% positive without permeabilization in the absence of manipulations involved in cell isolation (data not shown).

A summary of the results of these studies is shown in Table II. The C-terminal region of these proteins, after being released by limited proteolysis, has been demonstrated to permeabilize the cell membrane of bacteria and fungi (53). Endogenous peptide antibiotics have been investigated for therapeutic potential, especially in the setting of recent concerns over drug resistance (54). We have demonstrated not only the existence of a unique member of the Fc{gamma}RIIIa complex, but also have identified additional cell types that may express these cathelicidin molecules. We have demonstrated surface expression of the cathelin homologue on porcine PMN. We have not yet confirmed surface expression on porcine PAM or NK cells, but plan future characterization of these cell types. In our system, the proposed biological functions of the 15-kDa cathelin-like protein should take into account its localization to the cell surface and novel association with porcine Fc{gamma}RIIIa{alpha}. Perhaps the proregion of the cathelicidin associates with Fc{gamma}RIIIa{alpha} after processing of the C-terminal portion of the protein. Alternatively, the cathelin homologue may represent an antimicrobial protein specifically associated with porcine Fc{gamma}RIIIa{alpha} and may play some role in Fc{gamma}R-mediated function. It is possible that the 15-kDa cathelin homologue aids in functions of the Fc{gamma}R such as opsonization, phagocytosis, or cytotoxicity. The cathelicidin family of antibacterial peptides has recently been demonstrated to inhibit TNF-{alpha} expression through blockade of LPS binding CD14 (55). In addition, some of these antimicrobial proteins have been shown to direct the migration and activation of target cells by interacting with cell surface receptors and affecting release of various mediators of inflammation. These proteins may participate in host defense through their ability to induce expression of various target molecules involved in diverse biologic processes. It is interesting to note that the human cathelicidin hCAP-18 is processed to the antimicrobial peptide LL-37 by proteinase 3 (52), the target of c-antineutrophil cytoplasmic Abs associated with vasculitis. A recent study has demonstrated that porcine protegrin promotes the release of IL-1{beta} from LPS-stimulated monocytes via posttranslational modification (56). This provides additional evidence that the effects of these antimicrobial proteins may participate in acquired immunity and regulate the level of the inflammatory response. As the Fc{gamma}R serves to link humoral and cellular immunity, this novel association of the Fc{gamma}RIIIa{alpha} molecule and a cathelin-like protein may serve to link the innate and acquired branches of the immune response. Further studies of this novel association of antimicrobial proteins with Fc{gamma}RIIIa will certainly reveal new and exciting information relevant to the immune system. Characterization of the structure and function of this novel complex as it relates to host response to infectious agents and other systemic immune and autoimmune responses, will provide new insights into the role of the FcR and antimicrobial proteins in cellular biology.


View this table:
[in this window]
[in a new window]
  		Table II. Porcine cathelicidinsa

 


   Acknowledgments
 
We thank W. S. Lane, R. Robinson, J. Neveu, and E. Spooner (Harvard Microchemistry Facility, Harvard University, Cambridge, MA) for their expertise in the HPLC, mass spectrometry, and peptide mapping as well as S. Latshaw (Protein Sequence Laboratory, Finch University of Health Sciences/Chicago Medical School, Chicago, IL) for his skill in HPLC and protein sequencing. We also thank Drs. Tomas Ganz and Jishu Shi (University of California Los Angeles Medical Center, Los Angeles, CA); Dr. Peter Elsbach (New York University School of Medicine, New York, NY); and Dr. J. V. Ravetch (Member and Head, Laboratory of Biochemical Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY) for providing the antisera used in these experiments. Thanks also to Tony del Rosario and Remus Burchette for their assistance and maintenance of SPF miniature swine herds for this study.


   Footnotes
 
1 This work was supported in part by U.S. Public Health Service Grant RO1 CA52090 awarded by the National Cancer Institute, National Institutes of Health, U.S. Department of Health and Human Services. Back

2 Address correspondence and reprint requests to Dr. Yoon B. Kim, Department of Microbiology and Immunology, Finch University of Health Sciences/Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064. E-mail address: kimy{at}finchcms.edu Back

3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; SPF, specific pathogen-free; PAM, pulmonary alveolar macrophage; PPG, proprotegrin; DMP, dimethylpimeliminidate; PVDF, polyvinylidene difluoride; NRS, normal rabbit serum; PMAP, porcine myeloid antibacterial peptide. Back

Received for publication June 9, 2003. Accepted for publication November 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brooks, D., J. V. Ravetch. 1994. Fc receptor signaling. V. S. Gupta, ed. Mechanisms of Lymphocyte Activation and Immune Regulation 185. Plenum Press, New York.
  2. Ravetch, J. V., J. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.[Medline]
  3. Van de Winkel, J. G. J., P. J. A. Capel. 1993. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol. Today 14:215.[Medline]
  4. Mantzioris, B., M. Berger, W. Sewell, H. Zola. 1993. Expression of the Fc receptor for IgG (FcRII/CDw32) by human circulating T and B lymphocytes. J. Immunol. 150:5175.[Abstract]
  5. Unkeless, J. C., E. Scigliano, V. H. Freedman. 1988. Structure and function of human and murine receptors for IgG. Annu. Rev. Immunol. 6:251.[Medline]
  6. Unkeless, J. C.. 1989. Human Fc{gamma} receptors. Curr. Opin. Immunol. 2:63.[Medline]
  7. Van De Winkel, J. G. J., C. L. Anderson. 1991. Biology of human immunoglobulin G Fc receptors. J. Leukocyte Biol. 49:511.[Medline]
  8. Sandor, M., R. G. Lynch. 1993. Lymphocyte Fc receptors: the special case of T cells. Immunol. Today 14:227.[Medline]
  9. Lanier, L. L., J. J. Ruitenberg, J. H. Phillips. 1988. Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes. J. Immunol. 141:3478.[Abstract]
  10. Ravetch, J. V., B. Perussia. 1989. Alternative membrane forms of Fc{gamma}RIII (CD16) on human natural killer cells and neutrophils: cell type-specific expression of two genes that differ in single nucleotide substitutions. J. Exp. Med. 170:481.[Abstract/Free Full Text]
  11. Kurosaki, T., J. V. Ravetch. 1989. A single amino acid in the glycosyl phosphatidylinositol attachment domain determines the membrane topology of Fc{gamma}RIII. Nature 342:805.[Medline]
  12. Lanier, L. L., S. Cwirla, G. Yu, R. Testi, J. H. Phillips. 1989. Membrane anchoring of a human IgG Fc receptor (CD16) determined by a single amino acid. Science 246:1611.[Abstract/Free Full Text]
  13. Edberg, J. C., P. B. Redecha, J. E. Salmon, R. P. Kimberly. 1989. Human Fc{gamma}RIII (CD16): isoforms with distinct allelic expression, extracellular domains, and membrane linkages on polymorphonuclear and natural killer cells. J. Immunol. 143:1642.[Abstract]
  14. Ueda, E., T. Kinoshita, J. Nojima, K. Inoue, T. Kitani. 1989. Different membrane anchors of Fc{gamma}RIII (CD16) on K/NK-lymphocytes and neutrophils: protein- vs lipid-anchor. J. Immunol. 143:1274.[Abstract]
  15. Klaassen, R. J. L., W. H. Ouwehand, T. W. J. Huizinga, C. P. Engelfriet, A. E. G. K. Von Dem Borne. 1990. The Fc-receptor III of cultured human monocytes: structural similarity with FcRIII of natural killer cells and role in the extracellular lysis of sensitized erythrocytes. J. Immunol. 144:599.[Abstract]
  16. Selvaraj, P., O. Carpén, M. L. Hibbs, T. A. Springer. 1989. Natural killer cell and granulocyte Fc{gamma} receptor III (CD16) differ in membrane anchor and signal transduction. J. Immunol. 143:3283.[Abstract]
  17. Braakman, E., J. G. J. van de Winkel, B. A. van Krimpen, M. Jansze, R. L. H. Bolhuis. 1992. CD16 on human {gamma}{delta} T lymphocytes: expression, function and specificity for mouse IgG isotypes. Cell. Immunol. 143:97.[Medline]
  18. Farber, D. L., D. W. Sears. 1991. Rat CD16 is defined by a family of class III Fc{gamma} receptors requiring co-expression of heteroprotein subunits. J. Immunol. 146:4352.[Abstract]
  19. Letourneur, O., I. Kennedy, A. Brini, J. Ortaldo, J. O’Shea, J. Kinet. 1991. Characterization of the family of dimers associated with Fc receptors (Fc{epsilon}RI and Fc{gamma}RIII). J. Immunol. 147:2652.[Abstract/Free Full Text]
  20. Vivier, E., N. Rochet, J. P. Kochan, D. H. Presky, S. F. Schlossman, P. Anderson. 1991. Structural similarity between Fc receptors and T cell receptors: expression of the {gamma}-subunit of Fc{epsilon}RI in human T cells, natural killer cells and thymocytes. J. Immunol. 147:4263.[Abstract]
  21. Anderson, P., M. Caligiuri, J. Ritz, S. F. Schlossman. 1989. CD3-negative natural killer cells express {zeta} TCR as part of a novel molecular complex. Nature 341:159.[Medline]
  22. Lanier, L. L., G. Yu, J. H. Phillips. 1989. Co-association of CD3{zeta} with a receptor (CD16) for IgG Fc on human natural killer cells. Nature 342:803.[Medline]
  23. Anderson, P., M. Caligiuri, C. O’Brien, T. Manley, J. Ritz, S. F. Schlossman. 1990. Fc{gamma} receptor type III (CD16) is included in the {zeta} NK receptor complex expressed by human natural killer cells. Proc. Natl. Acad. Sci. USA 87:2274.[Abstract/Free Full Text]
  24. Kurosaki, T., I. Gander, J. V. Ravetch. 1991. A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions. Proc. Natl. Acad. Sci. USA 88:3837.[Abstract/Free Full Text]
  25. Kurosaki, T., I. Gander, U. Wirthmueller, J. V. Ravetch. 1992. The {beta} subunit of the Fc{epsilon}RI is associated with the Fc{gamma}RIII on mast cells. J. Exp. Med. 175:447.[Abstract/Free Full Text]
  26. Halloran, P. J., S. E. Sweeney, C. M. Strohmeier, Y. B. Kim. 1994. Molecular cloning and identification of the porcine cytolytic trigger molecule G7 as an Fc{gamma}RIII{alpha} (CD16) homologue. J. Immunol. 153:2631.[Abstract]
  27. Johnson, B. D., Y. B. Kim. 1990. Characterization of a monoclonal antibody enhancing porcine natural killer cell activity (PNK-E). Cell. Immunol. 125:107.[Medline]
  28. Johnson, B. D., W. G. Wierda, Y. B. Kim. 1991. Further characterization of PNK-E: a monoclonal antibody enhancing porcine natural killer cell activity. Cell. Immunol. 134:378.[Medline]
  29. Wierda, W. G., B. D. Johnson, M. E. Dato, Y. B. Kim. 1993. Induction of porcine granulocyte-mediated tumor cytotoxicity by two distinct monoclonal antibodies against lytic trigger molecules (PNK-E/G7). J. Immunol. 151:7117.[Abstract]
  30. Wierda, W. G., B. D. Johnson, M. D. Dato, Y. B. Kim. 1993. Two distinct porcine natural killer lytic trigger molecules as PNK-E/G7 molecular complex. Cell. Immunol. 146:270.[Medline]
  31. Halloran, P. J., S. E. Sweeney, Y. B. Kim. 1994. Biochemical characterization of the porcine Fc{gamma}RIII{alpha} homologue G7. Cell. Immunol. 158:400.[Medline]
  32. Sweeney, S. E., P. J. Halloran, Y. B. Kim. 1996. Identification of a unique porcine Fc{gamma}RIIIA{alpha} molecular complex. Cell. Immunol. 172:92.[Medline]
  33. Lenarcic, B., A. Ritonja, I. Dolenc. 1993. Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of the stefin family. FEBS Lett. 336:289.[Medline]
  34. Kim, Y. B., N. D. Huh, H. S. Koren, D. B. Amos. 1980. Natural killing (NK) and antibody-dependent cellular cytotoxicity (ADCC) in specific pathogen-free (SPF) miniature swine and germfree piglets. I. Comparison of NK and ADCC. J. Immunol. 125:755.[Medline]
  35. Rothlein, R., R. Gallily, Y. B. Kim. 1981. Development of alveolar macrophages in specific pathogen-free and germ-free Minnesota miniature swine. J. Reticuloendothel. Soc. 30:483.[Medline]
  36. Fernandez, J., M. DeMott, D. Atherton, S. C. Mische. 1992. Internal protein sequence analysis: enzymatic digestion for less than 10 µg of protein bound to polyvinylidene difluoride or nitrocellulose membranes. Anal. Biochem. 201:255.[Medline]
  37. Marchalonis, J. J.. 1969. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113:299.[Medline]
  38. Gersten, M. M., J. J. Marchalonis. 1978. A rapid, novel method for the solid-phase derivatization of IgG antibodies for immune-affinity chromatography. J. Immunol. Methods 24:305.[Medline]
  39. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[Medline]
  40. Zanetti, M., R. Gennaro, D. Romeo. 1995. Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain. FEBS Lett. 374:1.[Medline]
  41. Levy, O.. 1996. Antibiotic proteins of polymorphonuclear leukocytes. Eur. J. Haematol. 56:263.[Medline]
  42. Martin, E., T. Ganz, R. I. Lehrer. 1995. Defensins and other endogenous peptide antibiotics of vertebrates. J. Leukocyte Biol. 58:128.[Abstract]
  43. Lehrer, R., T. Ganz. 2002. Defensins of vertebrate animals. Curr. Opin. Immunol. 14:96.[Medline]
  44. Zhao, C., T. Ganz, R. I. Lehrer. 1995. The structure of porcine protegrin genes. FEBS Lett. 368:197.[Medline]
  45. Kokryakov, V. N., S. S. Harwig, E. A. Panyutich, A. A. Shevchenko, G. M. Aleshina, O. V. Shamova, H. A. Korneva, R. I. Lehrer. 1993. Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins. FEBS Lett. 327:231.[Medline]
  46. Gallo, R. L., M. Ono, T. Povsic. 1994. Syndecans, cell surface haparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide form wounds. Proc. Natl. Acad. Sci. USA 91:11035.[Abstract/Free Full Text]
  47. Zanetti, M., P. Storici, A. Tossi, M. Scocchi, R. Gennaro. 1994. Molecular cloning and chemical synthesis of a novel antibacterial peptide derived from pig myeloid cells. J. Biol. Chem. 269:7855.[Abstract/Free Full Text]
  48. Harwig, S. S. L., V. N. Kokyakov, K. M. Swiderek, G. M. Aleshina, C. Zhao, R. I. Lehrer. 1995. Prophenin-1, an exceptionally proline-rich antimicrobial peptide from porcine leukocytes. FEBS Lett. 362:65.[Medline]
  49. Zanetti, M., L. Litteri, G. Griffiths, R. Gennaro, D. Romeo. 1991. Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides. J. Immunol. 146:4295.[Abstract]
  50. Cowland, J., A. Johnsen, N. Borregaard. 1995. hCAP-18, a cathelin/probactenecin-like protein of human neutrophil specific granules. FEBS Lett. 368:173.[Medline]
  51. Weinrauch, Y., A. Foreman, C. Shu. 1995. Extracellular accumulation of potently microbicidal bactericidal/permeability-increasing protein and p15s in an evolving sterile rabbit peritoneal inflammatory exudate. J. Clin. Invest. 95:1916.
  52. Sorenson, O., P. Follin, A. Johnson, J. Calafat, G. Tjabringa, P. Hiemstra, N. Borregaard. 2001. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 97:3951.[Abstract/Free Full Text]
  53. Risso, A.. 2000. Leukocyte antimicrobial peptides: multifunctional effector molecules of innate immunity. J. Leukocyte Biol. 68<